structure activity relationships for inhibition of human 5a-reductases by polyphenols

8/9/2019 Structure activity relationships for inhibition of human 5a-reductases by polyphenols.

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Structureactivity relationships for inhibition of human

5a-reductases by polyphenols

Richard A. Hiipakka, Han-Zhong Zhang, Wei Dai, Qing Dai, Shutsung Liao*

Department of Biochemistry and Molecular Biology, The Ben May Institute for Cancer Research, and The Tang Center for

Herbal Medicine Research MC6027, University of Chicago, 5841 S. Maryland, Chicago, IL 60637, USA

Received 17 April 2001; accepted 1 August 2001

Abstract

The enzyme steroid 5a-reductase (EC 1.3.99.5) catalyzes the NADPH-dependent reduction of the double bond of a variety of 3-oxo-D4

steroids including the conversion of testosterone to 5a-dihydrotestosterone. In humans, 5a-reductase activity is critical for certain aspectsof male sexual differentiation, and may be involved in the development of benign prostatic hyperplasia, alopecia, hirsutism, and prostate

cancer. Certain natural products contain components that are inhibitors of 5a-reductase, such as the green tea catechin ()-

epigallocatechin gallate (EGCG). EGCG shows potent inhibition in cell-free but not in whole-cell assays of 5a-reductase. Replacement

of the gallate ester in EGCG with long-chain fatty acids produced potent 5a-reductase inhibitors that were active in both cell-free and

whole-cell assay systems. Other avonoids that were potent inhibitors of the type 1 5 a-reductase include myricetin, quercitin, baicalein,

and setin. Biochanin A, daidzein, genistein, and kaempferol were much better inhibitors of the type 2 than the type 1 isozyme. Several

other natural and synthetic polyphenolic compounds were more effective inhibitors of the type 1 than the type 2 isozyme, including

alizarin, anthrarobin, gossypol, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and octyl and dodecyl gallates. The presence of a

catechol group was characteristic of almost all inhibitors that showed selectivity for the type 1 isozyme of 5 a-reductase. Since some of

these compounds are consumed as part of the normal diet or in supplements, they have the potential to inhibit 5a-reductase activity, which

may be useful for the prevention or treatment of androgen-dependent disorders. However, these compounds also may adversely affect

male sexual differentiation. # 2002 Elsevier Science Inc. All rights reserved.

Keywords: 5a-Reductase; Polyphenols; Flavonoids; Green tea catechins; Epigallocatechin gallate; Prostate cancer

1. Introduction

The microsomal enzyme steroid 5a-reductase (EC1.3.99.5) catalyzes the NADPH-dependent reduction of

theD4,5 double bond of a variety of 3-oxo-D4 steroids [1,2],including the conversion of testosterone to 5a-dihydrotes-

tosterone, a process thought to amplify the androgenicresponse, perhaps because of the higher afnity of the

androgen receptor for 5a-dihydrotestosterone than fortestosterone [3]. Two different 5a-reductase isozymes have

been characterized in humans, monkeys, rats, and mice [47]. The two human isozymes share approximately 50%

sequence identity and have different biochemical proper-ties. For example, the type 1 isozyme has a broad basic pH

optimum and low afnity for testosterone (Km > 1 mM),while the type 2 isozyme has an acidic pH optimum and

high afnity for testosterone (Km < 10 nM) [8].Studies of mice with genetically engineered 5a-reduc-

tase gene knockouts, as well as investigations of natural5a-reductase deciencies in humans, have identied some

of the roles this enzyme plays in different biological

processes [7]. Female mice decient in the type 1 5a-reductase have impaired cervical ripening, leading todefective parturition [9,10]. This defect may be due to

impaired catabolism of cervical progesterone, which is

also a substrate for 5a-reductase. Uterine type 1 5a-reduc-tase is also important for fetal viability, because 5a-reduc-tase activity limits fetal exposure to toxic levels of 17b-

estradiol by competing for substrate with aromatase, which

catalyzes the synthesis of 17b-estradiol from testosterone[11]. In humans, activity of the type 2 isozyme is criticalfor differentiation of the prostate and male external geni-

talia [4,12,13]. Based on studies of individuals with

Biochemical Pharmacology 63 (2002) 11651176

0006-2952/02/$ see front matter # 2002 Elsevier Science Inc. All rights reserved.

PII: S 0 0 0 6 - 2 9 5 2 ( 0 2 ) 0 0 8 4 8 - 1

* Corresponding author. Tel.: 1-773-702-6999; fax: 1-773-834-1770.

E-mail address: [email protected] (S. Liao).

Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EC,

()-epicatechin; EGC, ()-epigallocatechin; ECG, ()-epicatechin gal-

late; EGCG, ()-epigallocatechin gallate.


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inherited deciencies in 5a-reductase activity as well as

laboratory studies of affected tissues, 5a-reductase may

also have a role in the development of a variety of humandisorders including benign prostatic hyperplasia [14], acne[15], alopecia [16,17], and hirsutism [18]. 5a-Reductase

also has been proposed to have a role in the development of

prostate cancer and possibly may be responsible for differ-ences in prostate cancer mortality among different racialgroups [19]. A common missense mutation (V89L) that

decreases the activity of the type 2 5a-reductase, is com-

mon among Asians, a group that has a lower mortality fromprostate cancer compared with AfricanAmerican men and

non-Hispanic whites [20]. Finasteride, a synthetic 5a-reductase inhibitor, is currently used to treat benign pro-

static hyperplasia [21] and alopecia [22], and it is alsobeing studied in clinical trials as a chemopreventative for

prostate cancer [23].Diet has an important role in modulating cancer inci-

dence and mortality, and differences in diet may explain

geographical differences in prostate cancer mortality

[24,25]. Since androgens regulate the growth and functionof the normal prostate and prostate cancer [26,27], dietarycomponents capable of altering this growth signaling path-

way in the prostate may affect prostate cancer development

and progression. We have shown that green tea containsphytochemicals called catechins that are inhibitors of 5a-reductase [28]. It is not known whether green tea catechins

modulate androgenic activity in vivo in humans, but rats

injected with the green tea catechin EGCG have a varietyof endocrine changes, including lower serum testosteroneconcentrations and smaller prostates than the controls [29].

Other natural product inhibitors of 5a-reductase that havebeen identied include polyunsaturated fatty acids, such asg-linolenic acid [30]; the macrocyclic ellagitannins,oenothein A and B [31,32]; the avonoids and lignans,

genistein, formononentin, biochanin A, daidzein, coumes-

trol, equol, and enterolactone [33]; the bisnaphthoquinone,impatienol [34]; and the isoprenylated avone and stilbene,

artocarpin and chlorophorin [35]. Since many of theseinhibitors of 5a-reductase are polyphenols, we have inves-

tigated the ability of a variety of natural and syntheticpolyphenols to inhibit 5a-reductase to determine what

structural elements are important for potent inhibition of

5a-reductase by this class of compounds in both the cell-free and whole-cell assay systems.

2. Materials and methods

2.1. Materials

[4-14C]-Testosterone (5060 mCi/mmol) was a productof Perkin-Elmer Life Sciences. Puried catechins, ()-

epicatechin (EC), ()-epigallocatechin (EGC), ()-epica-techin gallate (ECG), and EGCG, were isolated from green

tea (Camellia sinensis) in our laboratory as described [28].

Other chemicals either were purchased from Sigma or

Aldrich or were synthesized in our laboratory as described

below. Caffeic acid phenethyl ester was synthesized asdescribed [36]. A variety of semi-synthetic derivatives ofthe green tea catechin EGC were synthesized by esterifying

various aromatic and aliphatic acids to the 3-hydroxy

group of EGC (Fig. 1). EGC derivatives were synthesizedby acetylating all six hydroxyl groups of EGC using aceticanhydride in pyridine, followed by selective deacetylation

in Tris buffer at pH 8.2 to give the 3-monoacetate ( 2).

Silylation of the phenolic hydroxyls and subsequent dea-cetylation afforded pentasilylated epigallocatechin (4)

[37]. Aryl esters (5) of EGC were prepared by transester-ication of the 3-hydroxyl with the appropriate methyl

esters [38]. Aliphatic esters (7) of EGC were prepared byreaction with the requisite acids in the presence of dicy-

clohexylcarbodiimide and dimethylaminopyridine [39].Silyl protective groups were then removed from 5 and 7

with triethylamine trihydrouoride, providing the EGCanalogs. All compounds synthesized in our laboratory were

puried by silica gel chromatography and were character-ized by 1H-NMR. Spectroscopic data were consistent withtheir structure, e.g. EGC-myristoleate in CD3OD: 6.42

(2H, s, Harom), 5.92 (1H, d, J 2:28 Hz, Harom), 5.88

(1H, d, J 2:28 Hz, Harom), 5.365.28 (3H, m, CH=CH,H3), 4.92 (1H, s, H2), 2.82 (2H, t, J 7:48 Hz, CH2CO),2.00 (4H, m, CH2C=CCH2), 1.501.0 (14H, m, CH2),

0.90 (3H, t, J 7:1 Hz, CH3) ppm.

2.2. Expression of human 5a-reductase isozymes

and preparation of cell extracts

Cell lines expressing the different types of human 5a-

reductase were prepared as described previously [28]. Inbrief, cDNAs for the human types 1 and 2 5a-reductases

were isolated from human prostate cDNA libraries andsubcloned into the retroviral expression vector pMV7 [40],

and high titer stocks of viruses containing the types 1 and 25a-reductase cDNAs were generated using the packaging

cells BOSC 23 293 [41]. Rat 1A cells [42,43] were infected

with retrovirus, and cells containing integrated retroviruswere selected for resistance to G418 [44]. Cells expressingeither the type 1 or 2 human 5a-reductase were expanded

under G418 selection and, when conuent, were removedfrom plates by trypsinization. Cells were collected bycentrifugation (500 g for 10 min at 228), washed once withculture medium containing 10% fetal bovine serum,

quickly frozen on dry ice, and stored at 908 until used

in the preparation of a microsomal fraction containing5a-reductase.

Rat 1A cells were chosen for expression of 5a-reductase

based on their low background of 5a-reductase activity

(1.8 pmol/min/106 cells) compared with a variety of othercell types that were tested. 5a-Reductase activity (type 1 or2) in cells transduced with recombinant retroviruses con-

taining 5a-reductase cDNA was 28-fold greater (52 pmol/

1166 R.A. Hiipakka et al. / Biochemical Pharmacology 63 (2002) 11651176


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min/106 cells) than in cells transduced with control viruslacking 5a-reductase cDNA. Since the activity of therecombinant human 5a-reductase activity was so much

greater than endogenous rat 5a-reductase, our resultsshould reect characteristics of the exogenous humanand not the endogenous rat enzyme.

For the preparation of microsomes containing 5a-reduc-

tase, cells were thawed on ice and then homogenized in

10 vol. of 0.32 M sucrose containing 20 mM potassiumphosphate, pH 7, 1 mM EDTA, 0.1 mM phenylmethylsul-

fonyl uoride and 1 mg leupeptin/mL, using a Douncehomogenizer and then sonication. The homogenate was

centrifuged at 1000 g for 10 min at 48 and the pellet wasdiscarded. The supernatant was centrifuged at 100,000 g

for 60 min at 48, and the resulting pellet (microsomal

fraction) was resuspended at 1020 mg protein/mL in0.32 M sucrose, 20 mM potassium phosphate, pH 7,1 mM EDTA, using a Dounce homogenizer. This micro-

somal fraction was frozen on dry ice and stored at 908until used in assays of 5a-reductase. We assayed 5a-

reductase in various cellular fractions after differentialcentrifugation of cell extracts. Of the total 5a-reductase

activity in cell extracts, 7080% was present in the micro-somal fraction, 1020% in the nuclear fraction (1000 g

pellet), and 26% in the cytosolic fraction. 5a-Reductaseactivity in microsomes isolated from cells transduced with

virus containing 5a-reductase cDNA was 31-fold

(350 pmol/min per mg of protein; type 1) and 19-fold

(217 pmol/min per mg of protein; type 2) greater thanactivity (11.5 pmol/min per mg of protein) in microsomesfrom cells transduced with a control virus.

2.3. Assay of 5a-reductase

The cell-free assay was based on the measurement of

5a-dihydrotestosterone production from testosterone in thepresence of microsomes prepared from cells containing

either the type 1 or 2 human 5a-reductase. The assaymixture, in a nal volume of 0.25 mL, contained 3 mM

[4-14C]-testosterone, 0.1 mM NADPH, 100 mM potassium

phosphate, pH 7.0. Test compounds were prepared in wateror dimethyl sulfoxide, and 2.5 mL of appropriate dilutionswas added to a reaction mix prior to the addition of

enzyme. Controls contained similar concentrations of sol-vents. The reaction was started by the addition of 25 mL ofmicrosomes containing 25 mg of protein to 225 mL of anassay mixture at 378. The mixture was incubated at 378 for

1 hr, and the reaction was stopped by the addition of

0.5 mL of ethyl acetate and mixing for 1 min. Aftercentrifugation (10,000 g for 5 min at 228), the organicphase was removed, dried under vacuum, dissolved in

25 mL of ethyl acetate and applied to a silica gel 60

TLC plate, which was developed in a solvent systemconsisting of methylene chloride:ethyl acetate:methanol(85:15:3). Conversion of testosterone to 5a-reduced meta-

bolites was measured by scanning the TLC plate on an

Fig. 1. Synthesis scheme for various aromatic and aliphatic ester derivatives of EGC. Ac: acetate; TBDMS: tert-butyldimethylsilane; TBDMSCl: tertiary-

butyldimethylsilyl chloride; DCC: dicyclohexylcarbodiimide; DMAP: dimethylpyridine; THF: tetrahydrofuran; R H: aliphatic group; Ar: aromatic group.

R.A. Hiipakka et al. / Biochemical Pharmacology 63 (2002) 11651176 1167


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AMBIS radioanalytical scanner or scanning a phosphor

screen exposed to the TLC plate on a Storm 860 phos-

phoimager. 5a-Dihydrotestosterone was the predominantmetabolite (>95%), with little or no conversion of testos-terone to androstanediols, androstandione, 4-androstene-

dione, or other metabolites, and recovery of radioactivity

was from 90 to 95%.Inhibition of 5a-reductase activity was also measured inintact cells expressing either human type 1 or 2 5a-reduc-

tase. Cells were plated at 50,000 per well in a 24-well plate

in 1 mL of Dulbecco's modied Eagle's medium (DMEM)containing 50 U penicillin/mL, 50 mg streptomycin/mL,

and 10% fetal bovine serum and then placed in an incu-bator with 5% CO2 for 18 hr at 378. The medium was then

changed to 0.5 mL of serum-free DMEM, and 5 mL of testcompound in water or dimethyl sulfoxide was added. Cells

were incubated for 1 hr at 378 before the addition of[4-14C]-testosterone, at a nal concentration of 1.5 mM.

Then cells were incubated for 3 hr at 378, the medium wasremoved, and radioactive steroids were extracted with

ethyl acetate. The amounts of labeled testosterone and5a-dihydrotestosterone in extracts were determined byTLC and scanning the TLC plate as described above.

The concentration of test compound inhibiting the conver-

sion of testosterone to 5a-dihydrotestosterone by 50%(IC50) was determined by interpolation between appropriate

data points. 5a-Dihydrotestosterone was the predominant

metabolite (>95%), with little or no conversion of testos-

terone to androstanediols, androstandione, 4-androstene-dione, or other metabolites, and recovery of radioactivitywas from 85 to 90%.

2.4. HPLC analysis

The stability of EGCG at 378 in cell culture medium

(DMEM) equilibrated with 5% CO2 (pH 7.47.5) or in100 mM potassium phosphate buffer, pH 7.0, medium or

buffers that were used for whole-cell and cell-free assays of5a-reductase, respectively, was determined by monitoring

EGCG concentrations by HPLC using a Novapak (Waters)

C18 column (3:9 mm 150 mm) and isocratic elution at408 at a ow rate of 1 mL/min with acetonitrile:ethylacetate:0.05% phosphoric acid in water (12:2:86). EGCG

was detected by monitoring the absorbance at 275 nm.

3. Results

The structures of various compounds investigated in thisreport are shown in Figs. 2 and 3. A comparison of the

abilities of the four major green tea catechins, EC, EGC,ECG, and EGCG (Fig. 2), to inhibit the types 1 and 2

Fig. 2. Structures of flavonoids tested for inhibitory activity against 5a-reductase.



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isozymes of 5a-reductase in cell-free and whole-cellassays is presented in Table 1. Using a cell-free assay,

ECG and EGCG were better inhibitors of 5a-reductase

than were EC and EGC, and the type 1 isozyme was more

sensitive to these inhibitors than the type 2 isozyme. SinceECG and EGCG only differ structurally from EC and EGC

by the presence of a gallic acid ester on the 3-hydroxyl, the

gallate group is important for the enhanced ability of ECG

Fig. 3. Structures of polyphenols tested for inhibitory activity against 5a-reductase.



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and EGCG to inhibit 5a-reductase. These green tea cate-

chins had little inhibitory activity against 5a-reductase inwhole cells. The lack of activity in whole cells may be due

to an inability of these catechins to cross the cell membraneor to enzymatic or non-enzymatic changes in the structure

of these catechins in assays using whole-cell cultures. Thestability of EGCG in culture medium may be responsible,in large part, for the lower activity of EGCG in the cell

culture assay, since the half-life of EGCG in culture

medium and phosphate buffer used for whole-cell andcell-free 5a-reductase assays was 9:5 0:5 and74:7 6:4 min (mean SEM, N 3), respectively. The

stability of EGCG in aqueous solution is highly dependent

on pH [45,46], and the difference in pH between culturemedium (pH 7.5) and the phosphate buffer for cell-freeassays (pH 7.0) may be responsible, in part, for this 8-fold

difference in stability. The half-life of EGCG in phosphatebuffered saline, pH 7.5, was determined to be 21:2

2:0 min.Certain avonoids, including EGCG, produce hydrogen

peroxide in aqueous solutions at physiological pH, possi-

bly through a superoxide intermediate [47,48]. To deter-mine if these reactive oxygen species may have some role

in inhibition of 5a-reductase by EGCG, we added catalase(25250 mg/mL) or superoxide dismutase (0.55 mg/mL)

to assay mixtures containing EGCG. However, addition ofthese enzymes did not affect inhibition of 5a-reductase

type 1 or 2 by 20 or 100 mM EGCG. Therefore, peroxide

and superoxide do not appear to be responsible for inhibi-tion of 5a-reductase by EGCG.

We performed a kinetic analysis of the inhibition of type

1 5a-reductase by EGCG using the cell-free assay todetermine the mode of inhibition of EGCG [49]. EGCG

was a competitive inhibitor of the substrate NADPH and anon-competitive inhibitor of the substrate testosterone

based on double-reciprocal plots of the kinetic data (Fig. 4).To determine what structural features of the gallate

group of EGCG were important for inhibitory activityagainst 5a-reductase, and to determine whether structural

changes in or replacement of the gallate group could

enhance inhibitory activity in whole cells, a series of

EGC derivatives was synthesized and tested using thecell-free and whole-cell assays (Table 2). Modication

of the hydroxyl groups of the gallate ester by methylation

or replacement of gallic acid with various aromatic groupswithout phenolic groups did not improve inhibitory activityin either the cell-free or whole-cell assay. The most sig-

nicant structural change leading to enhanced activity in

the whole-cell assay was introduction of an aliphatic acidester in place of the gallic acid ester of EGCG. EGC

derivatives with long-chain aliphatic acids were better

inhibitors than derivatives with short-chain aliphatic acids,and derivatives with aliphatic acids with some degree ofunsaturation were better inhibitors than EGC derivatives

esteried with saturated aliphatic acids. EGC esteried toeither g-linolenic or myristoleic acid were potent inhibitors

of both 5a-reductases in whole cells with IC50 values of lessthan 15 mM. We have reported previously that certain

polyunsaturated fatty acids, such as g-linolenic acid, areinhibitors (IC50: 510 mM) of 5a-reductase [30]. Methyl

and cholesterol esters ofg-linolenic acid were not potentinhibitors of 5a-reductase (ic50 > 100 mM) in cell-free and

whole-cell assays (data not shown). Therefore, it is likely

that the enhanced inhibitory activity of EGC esteried to

Table 1

Inhibition of 5a-reductase isozymes by green tea catechinsa

Catechin 5a-Reductase

Cell-free assay IC50 (mM) Whole-cell assay IC50 (mM)

Type 1 Type 2 Type 1 Type 2

EC >100 (14) >100 (4) >100 (0) >100 (1)EGC >100 (15) >100 (3) >100 (15) >100 (1)

ECG 11 (100) 69 (83) >100 (0) >100 (0)

EGCG 15 (99) 74 (74) >100 (6) >100 (0)

aIC50: concentration (mM) of compound producing 50% inhibition of

5a-reductase activity. Values in parentheses are percent inhibition of 5a-

reductase activity in the presence of 100 mM concentration of the indicated

compound.

Fig. 4. Kinetic analysis of the inhibition of type 1 5a-reductase by EGCG.

Initial reaction velocities (V) were determined for different substrate (panel

A, testosterone; panel B, NADPH) concentrations and as a function of

EGCG concentration: 0 mM (&), 5 mM (^), and 10 mM (*) in panel A

and 0 mM (&), 20 mM (^), and 30 mM (*) in panel B.



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g-linolenic acid is due to the combined functionality of this

derivative and not simply due to hydrolysis of the ester

bond and release of free g-linolenic acid. Also, cellularmorphology, as determined by light microscopy, was notaltered when cells were incubated with EGC derivatives

containing g-linolenic or myristoleic acid esters; therefore,

inhibition of 5a-reductase in whole cells was not due togross changes in cell integrity.

To determine what other structural attributes were

important for inhibition of 5a-reductase by polyphenoliccompounds, we tested a variety of natural and syntheticpolyphenols for their ability to inhibit 5a-reductase iso-zymes in both the cell-free and whole-cell assays. Several

naturally occurring avonoids with structures related to the

tea catechins were tested (Fig. 2, Table 3). Four avonoids,myricetin, quercitin, baicalein, and setin, had marked

(ic50 < 100 mM) activity and were more active againstthe type 1 than the type 2 isozyme. The number and

position of B-ring hydroxyl groups appear to be importantfor inhibitory activity against the type 1 5a-reductase. The

avonols quercitin, myricetin, and setin, with a catechol

or pyrogallol conguration in the B-ring (Fig. 2), hadgreater inhibitory activity against the type 1 isozyme thanthe avonols chrysin, kaempferol, and morin that lack

hydroxyls in a catechol or pyrogallol conguration(Table 3). A comparison of the structures and inhibitory

activities of the avanols EC and EGC and the avonolsmyricetin and quercitin highlights the importance of a 2,3-

double bond and a 4-keto group in the C-ring for enhancedinhibitory activity. In contrast to quercitin, rutin, the 3-

rutinose glycoside of quercitin, was ineffective againsteither isozyme (ic50 > 100 mM). The inactivity of rutin

compared with quercitin may be due to the presence of the

bulky oligosaccharide rutinose causing steric hinderance or

to modication of the 3-hydroxy group. Taxifolin, a a-

vanone that is structurally similar to quercitin but lacking

the 2,3-double bond in the C-ring, was ineffective againsteither isozyme (ic50 > 100 mM). Biochanin A, kaemp-ferol, genistein, and daidzein were more effective inhibi-

tors of the type 2 than the type 1 isozyme. With the

exception of kaempferol, a avonol with a single B-ringhydroxyl, these type 2 inhibitors are isoavones with singlehydroxyls on the B-ring. The inhibitory effects of biocha-

nin A, genistein, and daidzein on 5a-reductase have beenreported previously [33]. When tested for inhibitory activ-ity in whole cells, most avonoids showed little or no

Tab1e 2

Inhibition of 5a-reductase by various density of EGCa

Group esterified to the 3-hydroxyl of EGC 5a-Reductase



H (EGC) 62 (61) >100 (30) >100 (15) >100 (1)Gallate (EGCG) 12 (99) 73 (76) >100 (11) >100 (5)

3,4,5-Trimethoxybenzoate 31 (94) >100 (20) >100 (12) >100 (7)

3,5-Dihydroxy-4-methylbenzoate 29 (93) 99 (51) NDb ND

4-Hydroxy-3,5-dimethoxybenzoate 29 (97) >100 (21) 43 (83) 62 (72)

Benzoate 48 (85) >100 (24) ND ND

Acetate (C2:0) >100 (35) >100 (8) >100 (8) >100 (9)

Caproate (C6:0) 47 (90) >100 (39) >100 (10) >100 (0)

Caprylate (C8:0) 30 (98) 78 (74) 58 (89) 72 (83)

Myristate (C14:0) 59 (95) 71 (84) 28 (97) 32 (98)

Myristoleate (C14:1) 20 (99) 76 (96) 7 (99) 8 (98)

Stearate (C18:0) >100 (31) >100 (0) 42 (90) 74 (81)

g-Linolenate (C18:3) 25 (97) 63 (93) 8 (99) 14 (98)

aIC50: concentration (mM) of compound producing 50% inhibition of 5a-reductase activity. Values in parentheses are percent inhibition of 5a-reductase

activity in the presence of 100 mM concentration of the indicated compound. EGC and EGCG are presented for comparison and are the natural compounds

isolated from green tea.b ND: not determined.

Table 3

Inhibition of 5a-reductase isozymes by various natural polyphenolsa

Polyphenol 5a-Reductase



Myricetin 23 (96) >100 (31) >100 (11) >100 (11)

Quercitin 23 (96) >400 (14) >100 (15) >100 (29)

Baicalein 29 (79) 99 (51) >100 (24) >100 (4)

Fisetin 57 (97) >100 (4) >100 (42) >400 (27)

Biochanin A 100 (50) 17 (74) 64 (64) 5 (93)

Daidzein >100 (3) 29 (69) >100 (13) 7 (89)

Kaempferol >100 (22) 12 (62) 79 (60) 20 (85)

Genistein >100 (16) 23 (76) >100 (22) 20 (89)

Morin >100 (6) >100 (33) NDb ND

Taxifolin >100 (5) >100 (5) ND ND

Chrysin >100 (2) >100 (1) ND ND

Rutin >100 (4) >100 (0) ND ND


5a-reductase activity. Values in parentheses are percent inhibition of

5a-reductase activity in the presence of 100 mM concentration of the

indicated compound.

b ND: not determined.



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activity against the type 1 isoenzyme, perhaps indicating

limited penetration of these polyhydroxy compounds

across the cell membrane or enzymatic or non-enzymaticchanges in the structure of these compounds in assaysusing whole-cell cultures. In contrast to the results with the

type 1 enzyme, four avonoids, biochanin A, daidzein,genistein, and kaempferol, had signicant inhibitory activ-ities against the type 2 isozyme in the whole-cell assay. Themost active of these, biochanin A and daidzein, have only

two and three free hydroxyl groups, respectively. These

avonoids may be active in whole cells because they maypenetrate cells easier than other avonoids that have more

hydroxyl groups. These avonoids also may be less sus-ceptible to modication in cell cultures.

Since avonoids that had catechol moieties were potentinhibitors of the type 1 5a-reductase, a variety of naturally

occurring and synthetic compounds that have catechol

groups as part of their structure were tested for inhibitoryactivity against 5a-reductase (Tables 4 and 5, Fig. 3).Sixteen compounds had IC50 values below 100 mM and

ve compounds had IC50 values below 10 mM. All weremore active against the type 1 than the type 2 isozyme.

Several of these compounds, including alizarin, anthrar-obin, dodecyl gallate, gossypol, octyl gallate, caffeic acid

phenethyl ester, and nordihydroguaiaretic acid also wereinhibitors of 5a-reductase in whole-cell assays with IC50values of less than 20 mM. In the whole-cell assay, anthrar-obin and alizarin were much more effective against the

type 1 than the type 2 isozyme, whereas the other ve

inhibitors were equally effective inhibitors of both iso-

zymes. Alizarin (1,2-dihydroxyanthraquinone) and pur-

purin (1,2,4-trihydroxyanthraquinone), which contain a

catechol group on an anthraquinone backbone, were potentinhibitors of 5a-reductase. The structural isomers of ali-zarin, anthraavic acid (2,6-dihydroxyanthraquinone),

anthrarun (1,5-dihydroxyanthraquinone), and quinizarin(1,4-dihydroxyanthraquinone), which do not have cate-chols in their structure, were weak inhibitors, highlightingagain the importance of catechols for inhibitory activity

against 5a-reductase. The p-quinone structure found in

alizarin may not be necessary for inhibitory activity, sinceanthrarobin (1,2,10-anthracenetriol) had inhibitory activity

Table 4

Inhibition of 5a-reductase isozymes by compounds containing catecholsa

Catechol 5a-Reductase



Anthrarobin 4 (99) 50 (97) 6 (91) >100 (31)Bromopyrogallol red 7 (98) 84 (58) NDb ND

Gossypol 7 (99) 21(99) 7 (100) 6 (99)

Pyrogallol red 15 (97) >100 (27) ND ND

Nordihydroguaiaretic acid 19 (99) 50 (80) 19 (99) 22 (99)

Caffeic acid phenethyl ester 25 (97) >100 (36) 8 (99) 7 (98)

Octyl gallate 27 (99) 58 (90) 7 (99) 18 (94)

Purpurogallin 30 (81) >100 (31) ND ND

Hydroxydopamine 42 (69) >100 (41) ND ND

Dodecyl gallate 43 (88) >100 (36) 3 (99) 7 (98)

Pyrocatechol violet 48 (85) >100 (47) ND ND

Pyrogallol 70 (60) >100 (28) >100 (7) >100 (15)

Caffeic acid >100 (13) >100 (8) ND ND

Esculetin >100 (7) >100 (13) ND ND

Ellagic acid >100 (7) >100 (9) ND ND

Catechol >100 (5) >100 (0) >100 (9) >100 (3)Methyl gallate >100 (5) >100 (3) >100 (0) >100 (0)

Propyl gallate >100 (0) >100 (0) >100 (5) >100 (0)

Fraxetin 100 (2) ND ND

aIC50: concentration (mM) of compound producing 50% inhibition of 5a-reductase activity. Values in parentheses are percent inhibition of 5a-reductase

activity in the presence of 100 mM concentration of the indicated compound.b ND: not determined.

Table 5

Inhibition of 5a-reductase isozymes by hydroxyanthraquinonesa

Anthraquinone 5a-Reductase



Purpurin 2 (95) >100 (20) NDb ND

Alizarin 3 (95) >100 (54) 6 (75) >100 (27)

Alizarin red S 30 (91) >100 (8) >100 (22) >100 (1)

Anthrarufin 40 (67) >100 (13) ND ND

Anthraflavic

acid

>100 (27) >100 (22) ND ND

Ouinizarin >100 (26) >100 (7) ND ND


5a-reductase activity. Values in parentheses are percent inhibition of 5a-

reductase activity in the presence of 100 mM concentration of the indicated

compound.

b ND: not determined.



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equivalent to that of alizarin (Tables 4 and 5). Alizarin red

S, which is structurally similar to alizarin, but has an

additional 3-sulfate group adjacent to the catechol, was1020 times less potent than alizarin (Table 5). Thecharged sulfate group on alizarin red S may interfere with

binding to 5a-reductase, a hydrophobic enzyme, as well as

interfere with the transport of this class of molecule acrossthe cell membrane. The difference in the activities ofcaffeic acid (ic50 > 100 mM) and caffeic acid phenethyl

ester (ic50 25 mM) (Table 4) may have a similar expla-

nation. Gossypol, a potent inhibitor in both whole-cell andcell-free assays, contains two catechol moieties. Both of

these groups could be contributing to the inhibitory activityof this compound. The methyl and propyl esters of gallic

acid were much less potent inhibitors of 5a-reductase thanthe octyl and dodecyl esters. The latter are more hydro-

phobic than the former and may interact more readily withmicrosomal 5a-reductase because of their hydrophobicnature. Dodecyl and octyl gallate were more potent inhi-

bitors in the whole-cell than in the cell-free assay (Table 4).

The long fatty acid esters on dodecyl and octyl gallate mayenhance uptake of these compounds in whole cells, andthese compounds may concentrate in cell membranes

leading to inhibition of 5a-reductase. Hydroxydopamine

(3,4,5-trihydroxyphenethylamine), which is structurallysimilar to the short-chain esters of gallic acid, but ispositively charged at physiological pH, had inhibitory

activity in the cell-free assay that was similar in potency

to that of dodecyl gallate. The compounds catechol (1,2-dihydroxybenzene), pyrogallol (1,2,3-trihydroxyben-zene), and gallic acid (3,4,5-trihydroxybenzoic acid), which

have catechols in their structure, hadweak inhibitory activityin both cell-free and whole-cell assay systems. Three dyes,bromopyrogallol red (5H,5HH-dibromopyrogallolsulfoneph-thalein), pyrocatechol violet (pyrocatecholsulfonephtha-

lein), and pyrogallol red (pyrogallolsulfonephthalein),

each containing catechol groups, were potent inhibitorsof 5a-reductase in the cell-free assay. In contrast to alirazin

red S, all three of these dyes are effective inhibitors, eventhough they contain a charged sulfate group. This differ-

ence in inhibitory potency may be because the sulfate inalirazin red S is adjacent to the catechol group, while the

sulfate group in the other dyes that are active inhibitors is

located some distance from the catechol group. Threenaturally occurring catechol-containing compounds, ellagicacid, a condensation product of two gallic acid molecules,

and the coumarins, esculetin (6,7-dihydroxycoumarin) andfraxetin (7,8-dihydroxy-6-methoxycoumarin), had little

activity (ic50 > 100 mM) in the cell-free assay.

4. Discussion

We performed this structureactivity relationship studyof 5a-reductase inhibitors in an effort to provide some

insight into the mechanism by which EGCG, a green tea

polyphenol, inhibits 5a-reductase, as well as to identify

other natural product inhibitors of this enzyme. Inhibition

studies were conducted using a cell-free assay, as well asassays with intact cells. The latter assay may provide someestimate of the potential of a particular compound to inhibit

5a-reductase activity in vivo. This study identied several

natural products that were inhibitors of 5a-reductase. Sincesomeof these compounds wereeffective on whole cells, theymay be capable of modulating the activity of 5a-reductase

in vivo. Activity in vivo will ultimately be dependent upon

attaining pharmacologically active levels of these agentsin target tissues, which will depend on absorption, distribu-

tion, metabolism, and excretion of the agents.There is the possibility that diets containing natural

inhibitors of 5a-reductase may adversely affect processesdependent upon 5a-reductase activity, such as male sexual

differentiation. Mutations in the type 2 5a-reductase genehave been linked to isolated cases of hypospadias, acommon developmental abnormality of the male repro-

ductive tract [50]. It also has been reported that a maternal

vegetarian diet during pregnancy is associated withhypospadias [51]. The authors of this study speculatedthat phytoestrogens in the maternal diet may have a

deleterious effect on the developing male reproductive

system. It also seems possible that natural or syntheticdietary components that are potent inhibitors of 5a-reduc-tase may have adverse effects on male sexual development

when consumed in high levels at critical periods during

pregnancy.A variety of natural and synthetic polyphenolic com-

pounds were found to be inhibitors of 5a-reductase. Many

of these compounds were better inhibitors of the type 1than the type 2 isozyme, while a few inhibited bothisozymes equally. Biochanin A, daidzein, genistein, andkaempferol were the only polyphenols tested that were

better inhibitors of the type 2 than the type 1 isozyme. The

rst three compounds are isoavones, while kaempferol isa avonol. These compounds are found in a variety of plant

dietary products including soybean-based products. Sincethe type 2 isozyme of 5a-reductase has a critical role in

prostate development and is the predominant isozymepresent in the adult human prostate [52], diets rich in these

particular compounds have the potential to affect the

development and function of the prostate by modulatingthe activity of 5a-reductase. Since excessive 5a-reductaseactivity has been proposed to be a possible contributing

factor in prostate cancer development or progression [19],the development and progression of prostate cancer may

also be affected by diets containing inhibitors of 5a-reductase. Certain natural dietary agents may have the

ability to act as prostate cancer chemopreventative agentsby modulating 5a-reductase activity. If some of these

compounds are active in vivo, they may be importantcandidates for prostate cancer chemopreventative agents,

either taken in pure or enriched formulations or as com-

ponents of natural dietary products. Differences in the



10/12

intake of dietary inhibitors of 5a-reductase also may be

responsible, in part, for geographical and racial differences

in prostate cancer mortality.A consistent observation in this study was that poly-

phenolic inhibitors of the type 1 5a-reductase had a

catechol in their structure. Flavonoids that were better

inhibitors of the type 2 than the type 1 5a-reductase,however, did not contain catechols. Although a catecholgroup was necessary for potent inhibition of the type 1

isozyme by polyphenols, it was not always sufcient. For

instance, the natural product ellagic acid contains twocatechol groups and was a weak (ic50 > 100 mM) inhibitor

of 5a-reductase. The proximity of the catechols in ellagicacid to other molecular groups may have steric effects, and

the highly constrained structure of ellagic acid may preventinter- and intra-molecular interactions necessary for inhi-

bition by catechol-containing compounds.Inhibition of the type 1 5a-reductase by EGCG, which

contains two separate catechol/pyrogallol groups in itsstructure, was competitive with the substrate NADPH.

Therefore, the catechol groups in EGCG may be interact-ing with amino acid residues important for binding of thiscofactor by 5a-reductase. Several studies, based upon

characterization of naturally occurring mutants [53,54],

site-directed mutagenesis [5557], and photoafnity label-ing [58] of 5a-reductase, have identied certain amino acidresidues that may have a role in substrate and cofactor

binding. NADPH-binding is altered by certain amino acid

changes in the carboxyl-terminal half of the protein, whilesubstrate (testosterone) or inhibitor (nasteride) binding isaffected predominantly by changes in the amino-terminal

half of the protein, although some changes in the carboxyl-terminal half also affect binding of substrate. Photoafnitylabeling of the rat type 1 5a-reductase with 2-azido NADP

modies a portion of the carboxyl-terminal half of the

protein that is conserved among human and rat 5a-reduc-

tase isoforms [58]. Since EGCG was a competitive inhi-bitor of NADPH, it may have inhibited the enzyme by

interactions with residues in the carboxyl-terminal portionof the protein. Although the inhibition of 5a-reductase

by EGCG was determined to be competitive, we haveobserved that inhibition of the type 1 5a-reductase by

100 mM EGCG could not be reversed by pelleting micro-

somes exposed to EGCG and then resuspending them innew reaction buffer without EGCG. EGCG either must bestrongly bound to microsomes or must permanently alter

5a-reductase causing irreversible inhibition. Inhibition of5a-reductase by EGCG also did not increase when micro-

somes containing the type 1 or 2 5a-reductase wereincubated with EGCG for 1560 min prior to the start

of the assay (unpublished observations).Natural polyphenols, such as EGCG and certain other

avonoids, have been shown to inhibit a variety of enzymes[59,60]. Three properties of these compounds that may be

responsible for their biological activity are their ability to

form complexes with certain metal ions, their anti-oxidant

and pro-oxidant activities, and their ability to form com-

plexes with proteins [59]. Given our current understanding

of the mechanism of 5a-reductase, it does not appear likelythat metal ion complexation or anti- or pro-oxidant activitywould be responsible for inhibition of 5a-reductase by

EGCG and other polyphenols. The tea catechins ECG and

EGCG will form precipitates with soybean lipoxygenaseand yeast alcohol dehydrogenase [61]. We also haveobserved that EGCG will rapidly precipitate certain pro-

teins, such as chicken egg white lysozyme (unpublished

observation). The basis for this precipitation activity hasnot been dened thoroughly, but it may be due to the ability

of certain polyphenols to form both numerous H-bondswith a protein, as well as unselective association of the

aromatic nuclei of a polyphenol with certain amino acids,especially prolines [59]. Types 1 and 2 human 5a-reduc-

tases contain 14 (5.4%) and 17 (6.7%) proline residues,respectively; hence, these enzymes are not proline-rich

proteins. Also, only ve of these proline residues are in thecarboxyl-terminal half of the protein containing the puta-

tive NADPH-binding site.Catechols can form o-quinones, which are known to

react covalently with both primary amines and sulfhydryls

[62]. There are reports that EGCG [63] and other green tea

catechins [64] react covalently with sulfhydryls. Both thetype 1 and 2 5a-reductases are inhibited by sulfhydrylmodifying agents, such as N-ethylmaleimide, 5,5H-dithio-

bis(2-nitrobenzoic acid), 2,2H-bispyridyldisulde, p-hydro-

xymercuribenzoate, and mercuric chloride (unpublishedobservation). However, inclusion of 0.110 mM dithio-threitol or b-mercaptoethanol in assays did not prevent

inhibition of 5a-reductase by EGCG (unpublished obser-vations). Therefore, it is unlikely that EGCG inhibited5a-reductase by covalently modifying essential sulfhydrylgroups.

Acknowledgments

This work was supported, in part, by grants from the

National Institute of Health and the Tang Foundation toS.L.

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structure activity relationships for inhibition of human 5a-reductases by polyphenols

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